A deep intronic splice-altering variant causes APECED syndrome through antisense oligonucleotide-targetable pseudoexon inclusion.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator () gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic variants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic variant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of -expressing primary patient monocyte-derived dendritic cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109-base pair frame-shifting pseudoexon. We also found low-level expression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic -expressing cell lines. Through protein modeling and transcriptomic analyses of -transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal transcript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing variant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.